P618: SECOND-GENERATION CD19 TARGETING TRI-SPECIFIC KILLER ENGAGER DRIVES ROBUST NK CELL FUNCTION AGAINST B CELL MALIGNANCIES
Authors: J. Miller, B. Kodal, P. Hinderlie, D. Vallera, G. Berk, V. Bachanova, M. Felices
Journal: HemaSphere (2022)

Background: Chronic Lymphocytic Leukemia (CLL) involves uncontrollable clonal expansion of CD5+/CD19+ B lymphocytes. While patients can sometimes coexist with CLL for years, eventually there is progression to bulky adenopathy and pancytopenia from marrow suppression requiring therapy. In recent years, targeted therapies, including utilization of the inhibitors for B cell receptor (BCR) signaling and for B cell lymphoma 2 (Bcl-2) protein, have been effective treatment options. However, these treatments are associated with limitations such as development of drug resistance and the lower treatment efficacy ratio in high-risk patients. Meanwhile, CAR-T cell immunotherapy failed to produce effective treatment outcomes against CLL, mostly due to defects in the effector T cells leading to product failures, as well as being associated with high levels of off-target cytotoxicity. Aims: Thus, approaches to CLL treatment are necessary and NK cell immunotherapy presents a viable and non-toxic option to this problem. Methods: Our group previously demonstrated the immunotherapeutic potential of utilizing first-generation CD19-targeting 161519 Tri-Specific Killer Engager (TriKE®) to drive natural killer (NK) cell cytotoxicity against B-cell malignancies. We have now produced a novel second-generation CD19 TriKE, 'CAM161519', utilizing a humanized camelid anti-CD16 VHH single domain antibody (sdAb) 'CAM16', a wild-type IL-15 component, and anti-CD19 tumor antigen scFv all linked via short peptide linkers. The TriKE primarily works by generating a cytolytic bridge between NK cells and CD19 expressing B cell malignancies while also providing an expansion and survival signal to the NK cells. Using flow-based assays and imaging based cytolytic assays we explored the capability of CAM161519 to target a variety of B cell lines (Raji, Daudi, and Nalm6) and primary B cell malignancies from patients with CLL and B cell acute lymphoblastic leukemia (B-ALL). Expanded NK cells were incubated with primary CLL targets from 6 different patients and no treatment (NT), rhIL-15 (30 nM), Rituximab (10 ug/mL), or CAM16159 (30 nM). NK cell degranulation and IFNg production was evaluated after 5 hours of co-culture (Figure). CLL killing was evaluated in a similar co-culture, but with a staining mix containing CD5 and CD19, to identify CLL tumor cells in order to determine the percent CLL targets killed normalized to incubation of CLL cells with NK cells alone (Figure Image). Results: The CAM161519 robustly activated NK cell degranulation (CD107a) and inflammatory cytokine production (IFNg) against B cell lines. The activation was specific as it was strongly reduced against CD19 knockout Nalm6 cells. The TriKE also induced specific NK cell proliferation. IncuCyte imaging based cytolytic assays demonstrated strong killing activity against Raji targets. When evaluating activity against primary targets, CAM161519 induced enhanced NK cell degranulation, cytokine production, and cytotoxicity using flow cytometry-based assays against primary CLL (Figure Image) and B-ALL targets when compared to IL-15 treatment. CAM161519 also rescued functionality of NK cells from CLL patients against their autologous CLL cells. Image:Summary/Conclusion: Taken together, these data are indicative of the potential for clinical translation of the CAM161519 TriKE in the CLL and B-ALL settings alone or in combination with cellular products.